This invention relates to peptide compositions that are useful as immunogens for the prevention of urinary tract infection. The peptide immunogen of the present invention comprises a FimH Adhesin Functional Site-Derived (FAFSD) target peptide and a helper T cell epitope (Th) having multiple class II MHC binding motifs. Optionally, the immunogenic peptide further comprises an invasin domain, which acts as a general immune stimulator. The helper T cell epitope and the invasin domain enable the host to generate an immune response specific against the FAFSD target peptide to prevent the adherence of Escherichia coli and other enterobacteria to the bladder mucosa and confer protection against urinary tract infection.
Urinary tract infection (UTI) is one of the most common disorders in women and children, resulting in 7-8 million physician and hospital visits per year at a cost of over $1 billion. It is estimated that by age 30, roughly 50 percent of women have had at least one incidence of UTI with 2-10 percent having recurrent UTI. Females are generally more prone to UTI because of their anatomy. Recent studies have shown that, on average, women who are 18-40 years old suffer 1-2 infections over a two year period. Older women are at risk with the incidence being as high as 30%. In most cases, UTI is not life threatening. Standard antibiotics usually offer quick relief, but when left untreated, the chronic recurrence of urinary tract infection can cause kidney damage and even death. A vaccine would reduce this toll but there has been little success in the development of a practicable vaccine for UTI (Service, Science 1997, 276:533).
Earlier attempts to produce a UTI vaccine using whole fimbriae were not successful in protecting against a broad range of disease-causing bacteria. Intact whole fimbriae do not elicit a strong antibody response to FimH adhesin. (Hanson and Brinton, Nature 1988, 332:265; Johnson, 1991; U.S. Pat. No. 4,454,117, Langermann et al, 1997). Vaccines comprising peptides of the major fimbrial protein FimA have been reported (Schmidt et al, J Exp Med, 1985; 161:705; U.S. Pat. No. 4,740,585). However, antibodies raised against FimA are not anti-adhesive and do not block attachment. Furthermore, vaccines based on the major components of the fimbriae contain variable sites and are expected to provide a narrow typespecific protection (Abraham et al, 1988).
Certain strains of Escherichia coli (E. coli) are the main cause of UTI. While many factors contribute to the initiation and progression of UTI, it is widely accepted that attachment of bacteria to tissue in the urinary tract is a first step in the initiation of active infection. A number of studies have pointed to a role for xe2x80x9cfimbriaexe2x80x9d or xe2x80x9cpilusxe2x80x9d organelles, the long filamentous proteinaceous appendages on the surface of E. coli, as the primary means by which the bacteria fasten onto urogenital tissue to establish an infection. Studies have shown that an overwhelming majority of the uropathogenic E. coli isolates express mannose-binding type 1 fimbriae (Johnson, Clin Microbiol Rev, 1991; 4:80).
Specifically, a cluster of eight to nine closely associated genes located in the bacterial chromosome are responsible for the biogenesis, assembly and function of type 1 fimbriae (Klemm and Christiansen, Mol Gen Genet, 1987; 208:439-445). Each type 1 fimbrial filament is 1-2 xcexcm long with a diameter of 7 nm. It is a heteropolymer comprising a major subunit FimA and three minor subunits FimF, FimG, and FimH. The FimA subunits constitute  greater than 95% of the total fimbrial proteins and are arranged in a tight right-handed helix forming a central axial hole (Klemm and Christiansen, 1987; Johnson, 1991).
More specifically, type 1 fimbriae has, as a minor component, the mannose-binding FimH adhesin which is serologically conserved throughout the Enterobacteriaceae genera (Abraham et al, Nature, 1988; 336:682). Immune electron microscopy has revealed FimH to be placed strategically at the distal fimbrial tips and along the fimbriae at various intervals (Abraham et al, J Bacteriol, 1987; 169:5530). The FimH molecules that are localized at the fimbrial tips appear to be complexed with FimG in a flexible fibrillum structure (Jones et al., Proc Natl Acad Sci USA, 1995; 92:2081). The presence of FimH is important for initiating bacterial infections in the urinary tract (Langermann et al, Science, 1997; 276:607). The mannose-binding domain of FimH is localized at the amino terminus region of FimH (Jones et al, 1995). The mannose-binding site is believed to promote attachment of the bacteria to D-mannose-containing receptors on the host mucosa cells. In fact, antibodies specific to residues of the amino terminus region of FimH inhibited attachment by type 1 fimbriated E. coli to human buccal cells and to the mouse bladder epithelium (Abraham et al, 1987; Thankavel et al, J Clin Invest, 1997; 100:1123). This indicates involvement of the mannose-binding domain of FimH in the adherence of fimbriae as a potential virulence determinant.
In light of the role played by FimH in promoting the adherence of E. coli, a more detailed structure-function study was conducted to map the functionally important domains within the FimH molecule. FimH is folded into two domains belonging to the all-beta class connected by a short linker. The full sequence of the FimH molecule is shown in FIG. 1. The NH2-terminal mannose-binding lectin domain comprises residues 1 to156, and the COOH-terminal fimbriae domain, which anchors the adhesin to the fimbriae, comprises residues 160 to 279. The lectin domain of FimH is an 11-stranded elongated xcex2 barrel with a jellyroll-like topology. A pocket capable of accommodating a monomannose unit is located at the tip of the domain, distal from the connection to the pilin domain (Choudhury et al, Science, 1999; 285:1061).
The identification the tip adhesins of FimH, as a key virulence factor provided a specific target for vaccine development. In contrast to the variability of the major fimbrial protein, FimH is conserved throughout the genera of the Enterobacteriaceae. This has implications for the development of broadly protective vaccines against UTI (Abraham et al, 1988).
Recent vaccine development has been focused against the presumed FimH virulence determinant and have been more successful. Antibodies were raised in mice to two forms of FimH protein: 1) a complex containing the periplasmic chaperone FimC bound to full-length FimH protein, and 2) a naturally occurring mannose-binding FimH truncate corresponding to two-thirds of the FimH amino terminal blocked the ability of uropathogenic E. coli to bind to cells of a human bladder epithelial cell line, and protected mice from infection in vivo (Langermann et al, 1997). In a more recent in vivo study, a vaccine based on the FimH-FimC chaperone complex immunogen protected cynomolgus monkeys from infection by an E. coli cystitis isolate (Langermann et al, J Infect Dis, 2000; 181:774).
Thankanel et al (1997) reported that a domain localized in the FimH adhesin of Escherichia coli Type 1 fimbriae is capable of receptor recognition. He further reported the use of a domain specific antibody to confer protection against urinary tract infection. In that report, mice actively immunized with sFimH1-25 peptide (see Table 3, SEQ ID NO:2) exhibited significantly lower levels of bacterial bladder colonization when challenged by type-1 fimbriated E. coli. As expected from the conservation of FimH sequence among type 1-fimbriated bacteria, broad immunological cross-reactivity was reported for type-1 fimbriated. Antibodies generated against a peptide-carrier protein immunogen wherein the peptide is SEQ ID NO:2 displayed significant cross-reactivity to type-1 frimbriated urinary tract isolates Klebsiella pneumoniae Cl111, K. pneumoniae Cl120, Enterobacter aerogenes, E. coli Cl115, E. coli Cl116, E. coli Cl118, E. coli Cl121, E. coli Cl123, E. coli Cl124, E. coli Cl5, and Serratia marescens Cl119 (Thankavel et al, J Clin.Invest., 1997, 100:1123)
However, in Thankavel""s study, the FimH adhesin peptides with built-in cysteine residues at both ends were conjugated to carrier proteins such as KLH through intermolecular crosslinking. It is known that carrier proteins are too complex for use in driving antibody responses to site-specific targets. The mass of the carrier molecule is much greater than that of the functionally important target peptide site. Consequently, the major immune response is directed to the carrier protein rather than to the target site of the peptide immunogen. Moreover, immunization with hapten-carrier conjugates frequently leads to carrier-induced immune suppression (Schutze et al., J Immunol, 1985, 135:2319).
Accordingly, a more suitable peptide-based is needed. It would be desirable to provide a synthetic Th-FAFSD peptide immunogen that generates a site-specific immune response without epitopic suppression by undesirable T cell responses. The peptide-based FimH immunogen should provoke an early and strong immune response in humans and animals to target FimH sites of functional importance for protective immunity without the adverse carrier-induced immune suppression. The peptide-based FimH immunogen should also be stable and well defined chemically with no need of elaborate downstream processing for ease of manufacture and quality control to avoid the need of an elaborate production plant.
Well-designed promiscuous Th/B cell epitope chimeric peptides capable of eliciting Th responses and resultant antibody responses in most members of a genetically diverse population expressing diverse MHC haplotypes have been reported. Th epitopes termed xe2x80x9cpromiscuous Thxe2x80x9d are known to evoke efficient site-specific T cell help and can impart immunogenicity to B cell epitopes that by themselves are poorly immunogenic. Such Th epitopic peptides react with helper T-cell receptors and the class II MHC molecules, in addition to antibody binding sites (Babbitt et al., Nature, 1985; 317:359) to stimulate a tightly focussed site-specific antibody response to target B cell site. Promiscuous Th comprise specific sequences derived from potent immunogens including measles virus F protein and hepatitis B virus surface antigen. Many known promiscuous Th (Table 1) have been shown to be effective in potentiating a poorly immunogenic peptide corresponding to the decapeptide hormone LHRH (U.S. Pat. No.5,759,551).
Potent Th epitopes range in size from approximately 15-30 amino acid residues in length, often share common structural features, and may contain specific landmark sequences. For example, a common feature is amphipathic helices, which are alpha-helical structures with hydrophobic amino acid residues dominating one face of the helix and with charged and polar residues dominating the surrounding faces (Cease et al., Proc. Natl. Acad. Sci. USA, 1987; 84: 4249). Th epitopes frequently contain additional primary amino acid patterns such as a Gly or charged residue followed by two to three hydrophobic residues, followed in turn by a charged or polar residue. This pattern defines what are called Rothbard sequences. Also, Th epitopes often obey the 1, 4, 5, 8 rule, where a positively charged residue is followed by hydrophobic residues at the fourth, fifth and eighth positions after the charged residue. Since all of these structures are composed of common hydrophobic, charged and polar amino acids, each structure can exist simultaneously within a single Th epitope (Partidos et al., J. Gen. Virol., 1991; 72:1293). Most, if not all, of the promiscuous T cell epitopes fit at least one of the periodicities described above.
These features may be incorporated into the designs of idealized artificial Th sites, including idealized combinatorial Th epitope libraries. For the design of combinatorial Th sites, lists of variable positions and preferred amino acids are available for MHC-binding motifs (Meister et al., Vaccine 1995; 13:581); and a method for producing combinatorial Th has been disclosed as structured synthetic antigen library peptides (WO 95/11998). Thus, the 1,4,5,8 rule can be applied together with combinatorial MHC-binding motifs in the assignment of positions for the invariant and degenerate sites of a combinatorial Th site and for the selection of residues for these sites, so as to vastly enlarge the range of immune responsiveness to an artificial Th. Examples of artificial idealized and idealized combinatorial library Th are shown in Table 2. See U.S. Pat. No. 6,025,468 and WO 95/11998.
A generalized immunostimulatory element of a domain of an invasin protein from the bacteria Yersinia spp has been reported (Brett et al., Eur J Immunol, 1993, 23: 1608-1614). The immune stimulatory property of invasin results from its capability to interact with the xcex21 integrin molecules present on T cells, particularly activated immune or memory T cells. The specific sequence for an invasin domain found to interact with the xcex21 integrins has been described by Brett et al (1993). A preferred embodiment of the invasin domain (Inv) for linkage to a promiscuous Th epitope has been previously described in U.S. Pat. No. 5,759,551 and is incorporated herein by reference. The said Inv domain has the sequence:
To be effective, a peptide immunogen must do more than merely evoke an anti-peptide response. An effective peptide immunogen must also evoke a functional immune response, i.e., the antibody produced must have immunological cross-reactivity to the authentic target. It is known that peptide immunogens generally do not to retain a preferred structure. Therefore, it is important in designing a peptide target site to introduce structural constraints. However, the imposed structural constraint must be able to mimic the conformation of the targeted epitope so that antibodies evoked will be cross-reactivities to that site on the authentic molecule (Moore, Chapter 2 in Synthetic Peptides A User""s guide, ed Grant, WH Freeman and Company: New York, 1992, pp 63-67).
Peptide immunogens have been designed employing promiscuous Th epitopes, the invasin domain, and with imposed structural constraint for a peptide-based vaccine for HIV (U.S. Pat. No. 6,090,388).
The present invention relates to a synthetic peptide immunogen capable of inducing antibodies against a FAFSD target peptide for the prevention of the adherence of E. coli and other enterobacteria to the bladder mucosa to confer protection against urinary tract infection. In particular, the peptide immunogen of this invention comprises one or more Th epitopes linked to a FAFSD target peptide, selected from the group consisting of SEQ ID NOS:3-8 and a crossreactive or immunologically functional analog of the FAFSD target peptide (Hereinafter referred to as xe2x80x9cFAFSD peptidexe2x80x9d). Optionally, the peptide immunogen further comprises an invasin domain (SEQ ID NO:72) as a general immune stimulator. These peptide immunogens of the present invention are effective, capable of inducing antibodies against FAFSD to prevent the adherence of E. coli and other enterobacteria to the bladder mucosa, thus conferring protection against urinary tract infection.
The peptide immunogen of this invention is represented by one of the following formula:
(A)n-(FAFSD peptide)-(B)o-(Th)m-X
or
(A)n-(Th)m-(B)o-(FAFSD peptide)-X
or
(FAFSD peptide)-(B)o-(Th)m-(A)n-X
or
(Th)m-(B)o-(FAFSD peptide)-(A)n-X
wherein
each A is independently an amino acid or an invasin domain;
each B is independently an amino acid or a linking group chosen from the group consisting of an amino acid, gly-gly, (xcex1, xcex5-N)lys, Pro-Pro-Xaa-Pro-Xaa-Pro (SEQ ID NO:73); NHCH(X)CH2SCH2COxe2x80x94, xe2x80x94NHCH(X)CH2SCH2CO(xcex5-N)Lys-, xe2x80x94NHCH(X)CH2S-succinimidyl(xcex5-N)Lys-, and xe2x80x94NHCH(X)CH2S-(succinimidyl)-;
each Th comprise an amino acid sequence that constitutes a helper T cell epitope, or an immune enhancing analog or segment thereof;
(FAFSD peptide) is a synthetic peptide B cell target site antigen selected from the group consisting of SEQ ID NOS:2-8 or an analog thereof that is cross-reactive with and immunologically functional for FimH of fimbriae of E. coli; 
X is an xcex1xe2x80x94COOH or xcex1xe2x80x94CONH2 of an amino acid;
n is from 0 to about 10;
m is from 1 to about 4; and
o is from 0 to about 10.
Another aspect of this invention provides a vaccine comprising an immunologically effective amount of a peptide composition in accordance with this invention and one or more pharmaceutically acceptable vaccine delivery vehicles. The vaccine when administered at an appropriate dosage will generate immunotherapeutic antibodies directed against the FAFSD peptide and prevent the adherence of E. coli and other enterobacteria to the bladder mucosa to confer protection against urinary tract infection.
A further aspect of the invention relates to a method administering the vaccine composition to a mammal for the prevention of the adherence of E. coli and other enterobacteria to the bladder mucosa to confer protection against urinary tract infection in a mammal.
Generally, the synthetic immunogenic peptide, therefore, comprise about 20 to about 100 amino acids comprising the following (1) a helper T cell (Th) epitope, (2) a FAFSD peptide selected from the group consisting of SEQ ID NOS: 3-8 and an immunologically effective analogue of thereof, (3) a spacer to separate the immunogenic domains, and optionally (4) an invasin domain (SEQ ID NO:72) as a general immunostimulatory site. The Th and FAFSD peptide of the peptide immunogen are separated by a spacer comprising one or more amino acids. The optional invasin domain may be inserted in any order into the peptide provided that the immunoreactivity of the target peptide is substantially preserved or that immunoreactivity to the FAFSD target peptide is generated.
Most preferrably, the peptide immunogen comprises (1) combining a FAFSD peptide with a selected promiscuous Th site to which the majority of a population of a mammal are responsive; or (2) combining a FAFSD peptide with an enlarged repertoire of Th through combinatorial chemistry to accommodate the variable immune responsiveness of a population, and (3) the stabilization of a desirable conformational feature of FAFSD peptide by cyclic constraint. Such peptide immunogens are preferred for their ability to generate a specific response to the FAFSD peptide with a broadly reactive Th response showing that the positioning of the epitopes and the cyclization is optimized.
It has been found that the peptide immunogen of the present invention, comprising a particular structural arrangement of a Th epitope alone or a Th epitope linked to an invasin domain with a target B cell site FAFSD peptide, wherein the functional site within the native structure of the FAFSD peptide is not disturbed, is effective in stimulating the production of antibodies as a vaccine against UTI.